AMPK signaling in cancer: dual roles, molecular mechanisms, and therapeutic potential.

AMP-Activated Protein Kinase Signaling Protects Oligodendrocytes that Restore Central Nervous System Functions in an Experimental Autoimmune Encephalomyelitis Model

Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular responses, including cell proliferation, differentiation, cell growth, and apoptosis. Because these responses are tightly related to cellular energy level, AMP-activated protein kinase (AMPK), which plays an essential role in energy homeostasis, has emerged as another key regulator. In the present study, we demonstrate a novel signal network between AMPK and MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK and three MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK. Under these conditions, inhibition of endogenous AMPK by expressing a dominant-negative form significantly potentiated ERK activation, indicating that glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116 cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent expression of dual-specificity phosphatase (DUSP) 1 & 2, which are negative regulators of ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK, further implying that a signal network between AMPK and ERK is a critical regulatory point in coupling the energy status of the cell to the regulation of cell survival.

Glucose-Based Regulation of miR-451/AMPK Signaling Depends on the OCT1 Transcription Factor

The hypothesis that AMP-activated protein kinase (AMPK) acts as the ‘metabolic governor’ has deservedly received considerable attention (Winder, 2001). Studies defining the signalling pathway are elegant, the level of effort almost Olympian in nature, and the implications mighty, in terms of both the science and the potential for clinical application. However, while new and exciting, the notion that a single pathway acts as a metabolic governor in working mammalian muscle overlooks a lot of what is known about metabolic regulation. Now, with the results of McConell et al. (2005) in this issue of The Journal of Physiology, it is time to reassess the relevance of the AMPK signalling pathway for the regulation of metabolism in working human muscle. The notion that AMPK or any other component of the signalling pathway acts as a metabolic governor begs for consideration of a definition of ‘metabolism’. Typically, metabolism is described as ‘the sum of all processes in a living organism’, and because these processes involve heat production, ‘metabolic rate’ is commonly defined as ‘the rate of heat production’ (Brooks et al. 2004). Proponents of the hypothesis of AMPK as a metabolic governor overlooked basic definitions of ‘metabolism’ and ‘metabolic rate’ and neither proposed nor provided data to show how changes in AMPK could affect ATP turnover or any other component of energy flux. To the contrary, changes in muscle AMPK level or signalling are likely to be the consequence of, rather than cause of, changes in muscle metabolic rate. Hence, in terms of applicability to the energetics of muscle contraction or any other metabolic consequence of physical exercise, the hypothesis lacked a guard against tests such as those imposed by McConell and colleagues, who uncoupled changes in metabolic rate and energy substrate partitioning from changes in AMPK signalling in muscles of men studied before and after short-term exercise training. While the AMPK hypothesis lacked a necessary component of metabolic regulation, the hypothesis did contain a component to explain energy substrate partitioning during exercise. However, by predicting that AMPK signalling could simultaneously increase working muscle glucose disposal and lipid oxidation, proponents of the hypothesis failed to appreciate classic results of indirect calorimetry on body respiratory exchange ratio (RER) or more recent results on muscle respiratory quotient (RQ) or blood glucose and fatty acid flux rates during exercise (Brooks et al. 2004). Whether from perspectives of the Randle Cycle (Randle, 1995) or Crossover Concept (Brooks & Mercier, 1994), up-regulation of the use of one energy source (e.g. glucose) should down-regulate use of other sources (e.g. lipid). The proposition that AMPK could signal increments in both glucose and lipid metabolism in working muscle was untenable as it is contrary to experience and predictions of models of metabolic regulation. And, in terms of the AMPK hypothesis itself, alarms should have gone off when it was observed that malonyl-CoA levels did not change in working human muscle and remained at a level well above the IC50 (Brooks et al. 2004). Factors that regulate muscle glycolytic and oxidative energy fluxes are powerful, well known and independent of AMPK signalling (Kushmerick & Conley, 2002). Hence, it is not surprising that the purported, but subtle, effects of AMPK signalling are overridden during contraction. But, when exercise stops RER and RQ decline, and relative lipid oxidation increases whereas carbohydrate oxidation decreases (Brooks et al. 2004). Can it then be that the impact of AMPK signalling emerges after exercise? As untenable as the AMPK signalling hypothesis is for muscle exercise, it would be a pity to now suddenly abandon it based on the study of McConnell et al. In retrospect, their efforts were necessary, but results were predictable based on results of many previous investigations. In hindsight, because consequences of AMPK signalling are easily overridden during muscle contraction when energy flux rate can increase one or two orders of magnitude, invocation of the pathway as a means to regulate metabolic rate and energy substrate partitioning during physical activity may have been misguided. It is likely that the real importance of AMPK signalling, or its absence, may emerge in other, near basal conditions such as recovery from physical activity, space flight, postprandial rest in healthy individuals or those suffering from obesity, type 2 diabetes, or other metabolic diseases where small, but persistent effects on energy substrate partitioning may have major long-term consequences. And finally, the paper of McConnell and colleagues reminds us that homeostatic regulation of high flux systems typically requires redundant controls. Such systems are seldom regulated by a single factor. Perhaps the influence of AMPK signalling on muscle metabolism during exercise is analogous to the role of hydrogen ion in the regulation of pulmonary minute ventilation and muscle blood flow. By its nature, general understanding in well-developed sciences such as physiology possesses a degree of inertia. Ultimately, science advances because outstanding hypotheses are articulated (e.g. Winder, 2001) and aggressively tested. McConnell and colleagues have tested aspects of the ‘metabolic governor’ hypothesis. Now, the actual role and conditions under which changes in AMPK affect the regulation of muscle metabolism need to be determined.

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Acute Lymphoblastic Leukemia (ALL) is the most common hematological malignancy and the main cause of cancer-related deaths in children. Therefore, search for novel treatment strategies is warranted. We identified AMP activated protein kinase (AMPK), a master regulator of bioenergetics, as a potential target for ALL therapy due to its effects on cell proliferation and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We demonstrated that treatment of NALM6 (Bp-ALL) and CEM (T-ALL) cells with AICAR, an AMPK activator, induced growth inhibition and apoptosis. Using metformin, another AMPK agonist, we found 40% growth inhibition, and up to five- and three-fold greater induction of apoptosis relative to controls in CEM and NALM6, respectively. Unexpectedly, rescue experiments with AMPK inhibitors Ara-A and compound-C (CC) failed to abrogate the cytotoxic effects induced by AICAR. When used alone, Ara-A induced 60% and 40% cell death in NALM6 and CEM cells, respectively, whereas CC induced 75- and 15-fold more apoptotic death relative to controls. To investigate the mechanism by which AMPK activation vs. inhibition induced apoptosis, we determined levels of P-AMPK (T172) and factors associated with the PI3K/Akt/mTOR and RAS/cRAF/Erk signaling pathways in NALM6 and CEM cells treated with either CC, AICAR, or in combination. Our data show that P-AMPK levels were decreased by CC and increased by AICAR. Additional Western blots demonstrated that these agents exerted opposite effects on Akt and RAS signaling. CC decreased P-Akt (S473) and activated the RAS pathway, while AICAR increased P-Akt. We showed that activation of Akt by AICAR down-regulated the RAS pathway via phosphorylation of cRAF (S259). P-mTOR (S2448) and P-4EBP1 (T70) exhibited a greater decrease in cells treated with CC + AICAR as compared to each agent alone. A significant decrease in P-Akt was also detected in cells treated with both agents vs. each drug alone. Together, our data indicate that AICAR and CC induce cell death in ALL cells by two different mechanisms mediated by AMPK: AICAR-activation of AMPK inhibited the RAS-dependent cell proliferation pathway, and CC-inhibition of AMPK by down-regulating the Akt cell survival pathway. These results suggest that alterations in AMPK signaling may regulate the cross-talk between the PI3K/Akt/mTOR and RAS/cRAF/Erk cascades and may dictate the fate of ALL cells by regulating apoptosis after exposure to agents targeting these pathways. Experiments co-targeting AMPK and Akt using AICAR and Akt-inhibitor X, respectively, induced synergistic growth inhibition in CEM (CI=0.90) and NALM6 (CI=0.85) cells compared to each drug alone. These findings provide a rationale for simultaneously targeting AMPK and key signaling factors associated with either PI3K/Akt/mTOR or RAS/cRAF/Erk pathways in ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5089.

Serum Starvation Regulates Autophagy of Human Periodontal Ligament Cells Through Reactive Oxygen Species Mediated Adenosine Monophosphate-Activated Protein Kinase/Mechanistic Target of RAPAMYCIN Axis.

Introduction: Trimetazidine (TMZ) is an anti-anginal drug that has been widely used in Europe and Asia. The TMZ can optimize energy metabolism via inhibition of long-chain 3-ketoacyl CoA thiolase (3-KAT) in the heart, with subsequent decrease in fatty acid oxidation and stimulation of glucose oxidation. However, the mechanism by which TMZ aids in cardioprotection against ischemic injury has not been characterized. Hypothesis: AMP-activated protein kinase (AMPK) is an energy sensor that control ATP supply from substrate metabolism and protect heart from energy stress. TMZ changes the cardiac AMP/ATP ratio via modulating fatty acid oxidation, thereby it may trigger AMPK signaling cascade that contribute to protection heart from ischemia/reperfusion (I/R) injury. Methods: The mouse in vivo regional ischemia and reperfusion by the ligation of the left anterior descending coronary artery (LAD) were used for determination of myocardial infarction. The infarct size was compared between C57BL/6J WT mice and AMPK kinase dead (KD) transgenic mice with or without TMZ treatment. The ex vivo working heart perfusion system was used to monitor the effect of TMZ on glucose oxidation and fatty acid oxidation in the heart. Results: TMZ treatment significantly stimulates cardiac AMPK and extracellular signal-regulated kinase (ERK) signaling pathways (p<0.05 vs. vehicle group). The administration of TMZ reduces myocardial infarction size in WT C57BL/6J hearts, the reduction of myocardial infarction size by TMZ in AMPK KD hearts was significantly impaired versus WT hearts (p<0.05). Intriguingly, the administration of ERK inhibitor, PD 98059, to AMPK KD mice abolished the cardioprotection of TMZ against I/R injury. The ex vivo working heart perfusion data demonstrated that TMZ treatment significantly activates AMPK signaling and modulating the substrate metabolism by shifting fatty acid oxidation to glucose oxidation during reperfusion, leading to reduction of oxidative stress in the I/R hearts. Conclusions: Both AMPK and ERK signaling pathways mediate the cardioprotection of TMZ against ischemic injury. The metabolic benefits of TMZ for angina patients could be due to the activation of energy sensor AMPK in the heart by TMZ administration.

Breast cancer (BC) is associated with type 2 diabetes mellitus (T2DM) and obesity. Glucagon-like peptide (GLP)-1 regulates post-prandial insulin secretion, satiety, and gastric emptying. Several GLP-1 analogs have been FDA-approved for the treatment of T2DM and obesity. Moreover, GLP-1 regulates various metabolic activities across different tissues by activating metabolic signaling pathways like adenosine monophosphate (AMP) activated protein kinase (AMPK), and AKT. Rewiring metabolic pathways is a recognized hallmark of cancer, regulated by several cancer-related pathways, including AKT and AMPK. As GLP-1 regulates AKT and AMPK, we hypothesized that it alters BC cells' metabolism, thus inhibiting proliferation. The effect of the GLP-1 analogs exendin-4 (Ex4) and liraglutide on viability, AMPK signaling and metabolism of BC cell lines were assessed. Viability of BC cells was evaluated using colony formation and MTT/XTT assays. Activation of AMPK and related signaling effects were evaluated using western blot. Metabolism effects were measured for glucose, lactate and ATP. Exendin-4 and liraglutide activated AMPK in a cAMP-dependent manner. Blocking Ex4-induced activation of AMPK by inhibition of AMPK restored cell viability. Interestingly, Ex4 and liraglutide reduced the levels of glycolytic metabolites and decreased ATP production, suggesting that GLP-1 analogs impair glycolysis. Notably, inhibiting AMPK reversed the decline in ATP levels, highlighting the role of AMPK in this process. These results establish a novel signaling pathway for GLP-1 in BC cells through cAMP and AMPK modulation affecting proliferation and metabolism. This study suggests that GLP-1 analogs should be considered for diabetic patients with BC.

BackgroundThe AMP-activated protein kinase (AMPK) is an evolutionarily conserved regulator of cellular energy homeostasis. As a nexus for transducing metabolic signals, AMPK cooperates with other energy-sensing pathways to modulate cellular responses to metabolic stressors. With metabolic reprogramming being a hallmark of cancer, the utility of agents targeting AMPK has received continued scrutiny and results have demonstrated conflicting effects of AMPK activation in tumorigenesis. Harnessing multi-omics datasets from human tumors, we seek to evaluate the seemingly pleiotropic, tissue-specific dependencies of AMPK signaling dysregulation.MethodsWe interrogated copy number variation and differential transcript expression of 92 AMPK pathway genes across 21 diverse cancers involving over 18,000 patients. Cox proportional hazards regression and receiver operating characteristic analyses were used to evaluate the prognostic significance of AMPK dysregulation on patient outcomes.ResultsA total of 24 and seven AMPK pathway genes were identified as having loss- or gain-of-function features. These genes exhibited tissue-type dependencies, where survival outcomes in glioma patients were most influenced by AMPK inactivation. Cox regression and log-rank tests revealed that the 24-AMPK-gene set could successfully stratify patients into high- and low-risk groups in glioma, sarcoma, breast and stomach cancers. The 24-AMPK-gene set could not only discriminate tumor from non-tumor samples, as confirmed by multidimensional scaling analyses, but is also independent of tumor, node and metastasis staging. AMPK inactivation is accompanied by the activation of multiple oncogenic pathways associated with cell adhesion, calcium signaling and extracellular matrix organization. Anomalous AMPK signaling converged on similar groups of transcriptional targets where a common set of transcription factors were identified to regulate these targets. We also demonstrated crosstalk between pro-catabolic AMPK signaling and two pro-anabolic pathways, mammalian target of rapamycin and peroxisome proliferator-activated receptors, where they act synergistically to influence tumor progression significantly.ConclusionGenetic and transcriptional aberrations in AMPK signaling have tissue-dependent pro- or anti-tumor impacts. Pan-cancer investigations on molecular changes of this pathway could uncover novel therapeutic targets and support risk stratification of patients in prospective trials.

The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway

No study has examined the response of skeletal muscle AMP-activated protein kinase (AMPK) signaling beyond the first 3 h after an acute exercise bout in humans. The purpose of this study was to assess AMPK signaling in human skeletal muscle immediately after a single bout of moderate-intensity endurance exercise and 3 and 24 h after the exercise bout. We examined AMPK signaling, and protein expression of AMPK alpha, ACC-beta, and nNOS mu in untrained individuals (four females and four males) during the 24-h period after a 60-min bout of moderate-intensity (63 +/- 1% VO2peak) cycling endurance exercise. AMPK alpha2 activity, AMPK alpha2 Thr172 phosphorylation, and ACC-beta Ser222 phosphorylation were increased immediately after exercise. These increases had all returned to basal levels at 3 and 24 h after exercise. Furthermore, an acute bout of exercise did not alter AMPK alpha1, AMPK alpha2, ACC-beta, or nNOS mu protein expression during the 24-h period after exercise. Although an acute bout of exercise elicits increases in AMPK signaling, this alone is not sufficient to induce sustained increases in either AMPK signaling or protein expression during the postexercise period.

Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and carcinogenesis in tubular epithelial cells, which may be largely attenuated by renin-angiotensin system blockade, implying the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

What is the central question of the study? Is the reduced signalling of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis in the heart, responsible for the reduced β-adrenergic responsiveness of the heart in obesity? What is the main finding and its importance? Inhibition of AMPK in isolated hearts prevented the reduced cardiac β-adrenergic responsiveness of obese rats, which was accompanied by reduced phosphorylation of AMPK, a proxy of AMPK activity. This suggests a direct functional link between β-adrenergic responsiveness and AMPK signalling in the heart, and it suggests that AMPK might be an important target to restore the β-adrenergic responsiveness in the heart in obesity. The obesity epidemic impacts heavily on cardiovascular health, in part owing to changes in cardiac metabolism. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in the heart and is regulated by β-adrenoceptors (β-ARs) in normal conditions. In obesity, chronic sympathetic overactivation leads to impaired cardiac β-AR responsiveness, although it is unclear whether AMPK signalling, downstream of β-ARs, contributes to this dysfunction. Therefore, we aimed to determine whether reduced AMPK signalling is responsible for the reduced β-AR responsiveness in obesity. In isolated hearts of lean and obese Zucker rats, we tested β-AR responsiveness to the β1 -AR agonist isoprenaline (ISO, 1×10-10 to 5×10-8 m) in the absence and presence of the AMPK inhibitor, compoundC (CC, 10μm). The β1 -AR expression and AMPK phosphorylation were assessed by Western blot. β-Adrenergic responsiveness was reduced in the hearts of obese rats (logEC50 of ISO-developed pressure dose-response curves: lean -8.53±0.13×10x mversus obese -8.35±0.10×10x m ; P<0.05 lean versus obese, n=6 per group). This difference was not apparent after AMPK inhibition (logEC50 of ISO-developed pressure curves: lean CC -8.19±0.12×10x mversus obese CC 8.17±0.13×10x m, P<0.05, n=6 per group). β1 -Adrenergic receptor expression and AMPK phosphorylation were reduced in hearts of obese rats (AMPK at Thr172 : lean 1.73±0.17a.u.versus lean CC 0.81±0.13a.u., and obese 1.18±0.09a.u.versus obese CC 0.81±0.16a.u., P<0.05, n=6 per group). Thus, a direct functional link between β-adrenergic responsiveness and AMPK signalling in the heart exists, and AMPK might be an important target to restore the reduced cardiac β-adrenergic responsiveness in obesity.

The adenosine monophosphate (AMP)-activated protein kinase (AMPK) sits at a central node in the regulation of energy metabolism and tumor progression. AMPK is best known to sense high cellular ADP or AMP levels, which indicate the depletion of energy stores. Previous studies have shown that the low expression of phosphorylated AMPK is associated with a poor prognosis of pancreatic cancer. In this study, we report that AMPK is also highly sensitive to extracellular matrix (ECM) stiffness. We found that AMPK is activated in cells when cultured under low ECM stiffness conditions and is functionally required for the metabolic switch induced by ECM stiffness. This regulation of AMPK requires the Hippo kinases but not LKB1/CaMKKβ. Hippo kinases directly phosphorylate AMPKα at Thr172 to activate AMPK at low ECM stiffness. Furthermore, we found AMPK activity is inhibited in patients with pancreatic ductal adenocarcinoma (PDAC) with high ECM stiffness and is associated with a poor survival outcome. The activation of Hippo kinases by ROCK inhibitor Y-27632 in combination with the mitochondrial inhibitor metformin synergistically activates AMPK and dramatically inhibits PDAC growth. Together, these findings establish a novel model for AMPK regulation by the mechanical properties of ECMs and provide a rationale for simultaneously targeting the ECM stiffness–Hippo kinases–AMPK signaling and low glucose–LKB1–AMPK signaling pathways as an effective therapeutic strategy against PDAC.